CASQ2 is a high capacity 2-Phenylmelatonin calcium binding protein localized to the SR in the vicinity of RyR. CASQ2 acts as an active calcium buffer that modulates local luminal calcium-dependent closure of RyRs. When intra-SR calcium levels decline, calcium free CASQ2 binds to the luminal side of RyRs, causing them to close. Cardiomyocytes with depleted CASQ2 levels behave similar to DEHP-treated cells��lower calcium transient amplitude, faster decay time, diminished SR load and increased spontaneous triggered activity. In CASQ2 null mice, reduced CASQ2 expression in nodal cells led to decreased SBR and sinoatrial bradycardia, similar to DEHP��s effect on hESC-CM shown here. When CASQ2 levels are reduced, the functional recovery of RyR release sites is accelerated making them prone to premature or spontaneous reactivation, as we often saw in DEHP-treated cells. SERCA is a main pump that transports calcium ions from the cytoplasm into the SR. Diminished SERCA levels are believed to be the main reason for a lack of frequency potentiation in failing hearts. Reduced SERCA activity can also explain longer times to reabsorb caffeine-induced calcium into the SR. The latter is common after application of caffeine, which enhances SR leak, making it harder for the SR to remove calcium from the cytosol, and ultimately resulting in a decreased rate of decay of calcium transients. Lastly, DEHP exposure clearly impacts the distribution of cnx-43, resulting in reduced gap junctional expression at the cell membrane. A reduction in membrane-associated cnx-43 is the most likely explanation for slowed conduction velocity and a higher activation threshold in DEHP-treated hESC-CM. The adverse effects of DEHP are unlikely to be mediated exclusively through gene expression changes ; 1-EBIO additional studies are necessary to pinpoint the mechanisms by which DEHP alters calcium handling and intercellular communication. The latter can include post-transcriptional regulation and alterations in protein expression, trafficking, stabilization and activity. The latter is the most common route of administration in the clinical setting, which largely avoids first pass metabolism. Our previously published studies revealed adverse effects of DEHP on cardiac electrical conduction and an arrhythmogenic phenotype in rat cardiomyocytes. However, additional studies are necessary to fully elucidate the exact pathways behind the adverse effects of phthalates, and their metabolites, on human cardiac muscle physiology.